Recombinant proteins have become very attractive candidates for the development of novel therapeutics. However, production of protein pharmaceuticals requires significant optimization of processes to obtain sufficient yields of specific biologically active polypeptides. It is well established that the expression of recombinant proteins in the cytoplasm of Escherichia coli, in particular mammalian recombinant proteins, frequently results in the formation of insoluble aggregates known as inclusion bodies. High cell density fermentation and purification of the recombinant protein from inclusion bodies of E. coli are two major bottlenecks for the cost effective production of therapeutic proteins (Panda, A. K, 2003, Adv. Biochem. Eng. Biotechnol., 85, 43). Similarly, for research purposes, where hundreds of proteins may need to be screened for various activities, the expression of soluble, active protein is desirable, thereby avoiding the step of first purifying inclusion bodies and then having to denature and refold protein each separately.
Examples of the many pharmaceutically important proteins that form insoluble inclusion bodies when expressed in the cytoplasmic space of E. coli include human Growth Hormone (hGH) (Patra, A. K. et al., 2000, Protein Expr. Purif, 18, 182; Khan, R. H, et al., 1998, Biotechnol. Prog., 14, 722), human Granulocyte-Colony Stimulating Factor (G-CSF) (Zaveckas, M. et al. 2007, J Chromatogr B Analyt Technol Biomed Life Sci. 852, 409; Lee, A. Y. et al., 2003, Biotechnol Lett., 25, 205,) and Interferon alpha (IFN-alpha; Valente, C. A. et al., 2006, Protein. Expr. Purif. 45, 226). Furthermore, the immunoglobulin domains of antibodies and their fragments, including domain antibody fragments (dAb), Fv fragments, single-chain Fv fragments (scFv), Fab fragments, Fab′2 fragments, and many non-antibody proteins (such as FnIII domains) generally form inclusion bodies upon expression in the cytoplasm of bacterial hosts (Kou, G., et al., 2007, Protein Expr Purif. 52, 131; Cao, P., et al. 2006, Appl Microbiol Biotechnol., 73, 151; Chen, L. H et al., 2006, Protein Expr Purif.; 46, 495).
Human proteins typically fold using a hydrophobic core comprising a large number of hydrophobic amino acids. Research has shown that proteins can aggregate and form inclusion bodies, especially when genes from one organism are expressed in another expression host, such that the protein's native binding partners are absent, so that folding help is unavailable and hydrophobic patches remain exposed. This is especially true when large evolutionary distances are crossed: a cDNA isolated from a eukaryote for example, when expressed as a recombinant gene in a prokaryote, has a high risk of aggregating and forming an inclusion body. While the cDNA may properly code for a translatable mRNA, the protein that results will emerge in a foreign microenvironment. This often results in misfolded, inactive protein that generally accumulates as aggregates if the concentration is high enough. Other effectors, such as the internal microenvironment of a prokaryotic cell (pH, osmolarity) may differ from that of the original source of the gene and affect protein folding. Mechanisms for folding a protein may also be host-dependent and thus be absent in a heterologous host, and hydrophobic residues that normally would remain buried as part of the hydrophobic core instead remain exposed and available for interaction with hydrophobic sites on other proteins. Processing systems for the cleavage and removal of internal peptides of the expressed protein may also be absent in bacteria. In addition, the fine controls that may keep the concentration of a protein low will also be missing in a prokaryotic cell, and over-expression can result in filling a cell with protein that, even if it were properly folded, would precipitate by saturating its environment.
The recovery of biologically active products from the aggregated state found in inclusion bodies is typically accomplished by unfolding with chaotropic agents or acids, followed by dilution or dialysis into optimized refolding buffers. However, many polypeptides (especially structurally complex oligomeric proteins and those containing multiple disulfide bonds) do not easily adopt an active conformation following chemical denaturation. Small changes in primary structure can affect solubility, presumably by altering folding pathways (Mitraki, A. et al. (1989) Bio/Technology 7, 690; Baneyx, F, et. al. 2004 Nat Biotechnol, 22, 1399; Ventura, S. 2005 Microb Cell Fact, 4, 11). In order to reduce the formation of insoluble aggregates during high-density fermentation, some groups have linked heterologous fusion proteins to the protein of interest. Examples of such fusion sequences are Glutathione-S-Transferase (GST), Protein Disulfide Isomerase (PDI), Thioredoxin (TRX), Maltose Binding Protein (MBP), His6 tag (SEQ ID NO: 1), Chitin Binding Domain (CBD) and Cellulose Binding Domain (CBD) (Sahadev, S. et al. 2007, Mol. Cell. Biochem.; Dysom, M. R. et al. 2004, BMC Biotechnol, 14, 32). In summary, these approaches were found to be protein-specific, as they do not work for all proteins.
While various fusion proteins have been designed to improve folding, chemical PEGylation of proteins has also been reported to enhance protein solubility, reduce aggregation, reduce immunogenicity, and reduce proteolysis. Nonetheless, the proper folding of overproduced polypeptides remains problematic within the highly concentrated and viscous environment of the cell cytoplasm, where aggregation occurs in a concentration-dependent manner. Another approach for the expression of mammalian proteins in bacterial hosts avoids leader peptides and expresses the active protein directly in the cytoplasm of the host. However, this process tends to result in aggregation and inclusion body formation.
One widely used approach for the expression of mammalian proteins in active form in bacteria is to direct the protein into the non-reducing environment of the periplasmic space of bacterial hosts such as E. coli, typically using signal- or leader-peptides to direct secretion. Secretion into the periplasm (and rarely into the media) appears to mimic the native eukaryotic process of protein secretion, folding and disulfide formation and often results in active protein. This approach has many profound drawbacks. The periplasm tends to give low yields; the process is generally limited to smaller proteins; the process tends to be protein-specific; and also that the procedures for extracting periplasmic proteins are not as robust as extraction from the cytoplasm, which contributes to low yields. For these reasons, expression of proteins in the periplasm of bacteria is not applicable to most pharmaceutical proteins, which are typically commercially expressed in yeast or mammalian cell lines.
Another approach that has been tried to make mammalian proteins express in the cytoplasm of bacteria without forming inclusion bodies is to over-express folding-helper proteins, like the molecular chaperones which play a role in a wide range of biotechnological applications (Mogk et al. 2002 Chembiochem 3, 807). To date, several different families of chaperones have been reported. All are characterized by their ability to bind unfolded or partially unfolded proteins and release correctly folded proteins into the cytoplasm of bacteria. A well-characterized example is the heat-shock family of proteins (Hsp), which are designated according to their relative molecular weight, as described by Buchner, J., Faseb J. 1996 10, 10 and by Beissinger, M. and Buchner, 1998. J. Biol. Chem. 379, 245. While many bacterial and eukaryotic chaperonins have been tried for over-expression of proteins in bacteria and to a lesser extent mammalian cells, this approach has generally had little or no effect and this is less often practiced for expression optimization.
Many therapeutic proteins suffer from a number of drawbacks including short half-life, high serum clearance, and high immunogenicity. Sustained-release, or depot, formulations of protein therapeutics offer a strategy to decrease the frequency of protein injections and thus reduce the chance of undesired immune response and also increase patient compliance. The active pharmaceutical ingredient (API) is typically encapsulated in a matrix made of a biodegradable polymer, which then allows slow release of the API upon administration to a patient. Drugs administered as sustained-release formulations may also exhibit a less dramatic burst in bioavailability following injection (‘bolus effect’) compared to drug alone. This is very important as all drugs have a defined therapeutic window. At concentrations higher than the therapeutic window, the drug is toxic, whereas at concentrations below the therapeutic window, the drug no longer exhibits its biological or therapeutic effect. Depot formulations can provide a way to increase the period a drug is present in this therapeutic window.
Once the drug is released, however, the half-life of peptide or protein therapeutics remains relatively short. Improvements to therapeutic properties of proteins, in particular plasma clearance and immunogenicity, by attaching non-proteinaceous polymers to the proteins, have previously been described (Kochendoerfer, G. (2003) Expert Opin Biol Ther, 3: 1253-61), (Greenwald, R. B., et al. (2003) Adv Drug Deliv Rev, 55: 217-50), (Harris, J. M., et al. (2003) Nat Rev Drug Discov, 2: 214-21).
Therapeutic antibodies are typically administered bi-weekly or monthly but currently similar regimens for other therapeutic proteins still need to be developed. Recently, PLGA microspheres containing insulin modified with PEG (5 kDa) have been described as a controlled release formulation, but due to the short half-life of insulin, weekly injection intervals may still be required (Hinds, K. D., et al. (2005) J Control Release, 104:447-60).